Process for the separation of enantiomers and enantiopure reagent

ABSTRACT

Process for the separation of enantiomers comprising at least one free functional group, in which process a reagent based on an enantiopure amino acid is reacted in basic medium with a mixture comprising enantiomers.

[0001] The invention relates to a process for the separation ofenantiomers and to reagents based on an enantiopure amino acid which canbe used in the separation of enantiomers.

[0002] The separation of enantiomers is a matter of great importance inthe pharmaceutical, chemical and biotechnology industries. This isbecause the two enantiomers of a chemical substance with an identicalcomposition can have radically different biological activities. It isthus desirable to have available separation reagents and techniqueswhich make it possible to separate the enantiomers and to analyse theenantiomeric purity of pharmaceutical, chemical and biotechnologyproducts.

[0003] An article by Marfey, P., (Carlsberg Res. Comm., 49, 1984,591-596) describes a process for the separation of enantiomers byRP-HPLC. According to this known process,1-fluoro-2,4-dinitrophenyl-5-L-alaninamide is used as reagent for thederivatization of amino acids. Other similar processes are also known.However, the process and reagent which are described by Marfey and theother processes and reagents exhibit numerous disadvantages.

[0004] The derivative obtained in the reaction of the amino acid withthe reagent has to be isolated by successive neutralization, drying,redissolution and filtration operations. These operations take a greatdeal of time and are therefore not very advantageous in an industrialapplication. Furthermore, there is a risk, in cases of analyticalapplications, of errors in the analytical results caused by differingsolubility of the diastereomeric derivatives in the redissolutionsolvent. When quantitative analyses are carried out using UVspectrometry, difficulties due to differences in absorption coefficientof the diastereomeric derivatives are encountered in the known process.Finally, the high cost of the reagent renders it desirable to findalternatives.

[0005] The invention is targetted at overcoming these problems.

[0006] The invention consequently relates to a process for theseparation of enantiomers comprising at least one free functional group,in which

[0007] (a) a mixture comprising the enantiomers is reacted in basicmedium with a reagent based on an enantiopure amino acid, in whichreagent at least one amino group of the amino acid carries an activatinggroup, in order to form an active precursor of an isocyanate group, andin which reagent at least one carboxyl group of the amino acid issubstituted, and

[0008] (b) the mixture of diastereomers obtained is subjected to aseparation operation.

[0009] It has been found, surprisingly, that the process according tothe invention makes it possible to obtain good results with regard tothe separation of enantiomers comprising at least one free functionalgroup, in particular in quantitative analytical applications. Theprocess according to the invention makes possible rapid derivatizationand rapid separation of enantiomers under flexible and economicalconditions.

[0010] The invention also relates to a reagent based on an enantiopureamino acid in which at least one amino group of the amino acid carriesan activating group in order to form an active precursor of anisocyanate group and in which at least one carboxyl group of the aminoacid is substituted.

[0011] The term “amino acid” is understood to denote, for the purposesof the present invention, any compound comprising at least one NH₂ groupand at least one carboxyl group. The amino acids used in the presentinvention are chiral amino acids comprising at least one asymmetriccarbon. Use may be made of any chiral amino acid well known in itself ofnatural or synthetic origin.

[0012] Examples of reagents according to the invention are based, forexample, on the following natural amino acids:

[0013] alanine, valine, norvaline, leucine, norleucine, isoleucine,serine, isoserine, homoserine, threonine, allothreonine, methionine,ethionine, glutamic acid, aspartic acid, asparagine, cysteine, cystine,phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine,ornithine, glutamine and citrulline.

[0014] Unnatural enantiomers can also be used.

[0015] Examples of amino acids of synthetic origin which can be used asbasis for the reagent according to the invention comprise, for example,the following amino acids: (1-naphthyl)alanine, (2-naphthyl)alanine,homophenylalanine, (4-chlorophenyl)alanine, (4-fluorophenyl)alanine,(3-pyridyl)alanine, phenylglycine, diaminopimelic acid(2,6-diaminoheptane-1,7-dioic acid), 2-aminobutyric acid,2-aminotetralin-2-carboxylic acid, erythro-β-methylphenylalanine,threo-β-methylphenylalanine, (2-methoxyphenyl)alanine,1-amino-5-hydroxyindan-2-carboxylic acid, 2-aminoheptane-1,7-dioic acid,(2,6-dimethyl-4-hydroxyphenyl)alanine, erythro-β-methyltyrosine orthreo-β-methyltyrosine.

[0016] The term “enantiopure amino acid” is understood to denote achiral amino acid composed essentially of one enantiomer. Theenantiomeric excess (ee) is defined: ee (%)=100(x₁−x₂)/(x₁+x₂) withx₁>x₂; x₁ and x₂ represent the content of enantiomer 1 or 2 respectivelyin the mixture.

[0017] Use is generally made of an enantiopure amino acid with anenantiomeric excess of greater than or equal to 99%. Preference is givento an enantiopure amino acid with an enantiomeric excess of greater thanor equal to 99.5%. In a particularly preferred way, use is made of anenantiopure amino acid with an enantiomeric excess of greater than orequal to 99.9%.

[0018] Any enantiopure amino acid can be used as basis for the reagentaccording to the invention. The enantiopure amino acid is preferablyselected from the abovenamed amino acids of natural or synthetic origin.Amino acids comprising at least one aromatic nucleus, such as, forexample, phenylalanine or its derivatives, are particularly well suitedas enantiopure amino acid. In a particularly preferred way, theenantiopure amino acid is selected from phenylalanine,(1-naphthyl)alanine, (2-naphthyl)alanine or α- or β-tryptophan((2-indolyl)alanine or (3-indolyl)alanine), which are optionallysubstituted.

[0019] In the reagent according to the invention, at least one aminogroup of the enantiopure amino acid carries an activating group in orderto form an active precursor of an isocyanate group.

[0020] The term “active precursor of an isocyanate group” is understoodto denote any precursor which, when it is employed in a solvent whichcan be used in the process according to the invention with 1 equivalentof phenylalanine in the presence of 1 equivalent of base, reacts at atemperature of less than or equal to 35° C. essentially completely in aperiod of time of less than or equal to 30 min to form the correspondingurea. The reactive precursor preferably releases the isocyanate group ata temperature of less than or equal to 30° C. in a period of time ofless than or equal to 15 min. In a very particular preferred way, thereactive precursor releases the isocyanate group at room temperature ina period of time of less than or equal to 10 min. Test conditions whichcan be used to determine the active precursor are described, forexample, in Example 3 below.

[0021] The activating group is generally composed of a carbonylderivative bonded to an electronegative substituent. Use may be made,for example, as activating group, of an aryloxycarbonyl,heteroaryloxycarbonyl, 1,3-imidazolyl-N-carbonyl or1,2,4-triazolyl-N-carbonyl group. The aryloxycarbonyl groups which arewell suited include those which carry at least one −I, −M substituent onan aromatic nucleus. An −I, −M substituent is a group which has anegative inductive effect and negative resonance effect as defined in J.March, Advanced Organic Chemistry, 4th Ed., 1992, p. 17-19, 273-275. The−I, −M substituents include, for example, —NO₂, —SO₂R, —SO₂OR, —NR₃ ⁺and SR₂ ⁺. The substituents are preferably found at at least one of the2, 4 or 6 positions of the aromatic nucleus or at positions analogous tothe 2 or 4 positions in condensed aromatic systems. It is preferable touse an aryloxycarbonyl activating group which carries at least one nitrosubstituent on the aromatic nucleus. The (4-nitrophenyloxy)carbonylgroup is particularly preferred.

[0022] In an alternative form, the electronegative substituent comprisesan −I substituent (negative inductive effect) independently of theresonance effect (M) as they have been defined above. In thisalternative form, the activating group is preferably an aryloxycarbonylgroup carrying at least one −I substituent. The −I substituent ispreferably found at the positions which were taught above for the −I, −Msubstituents. Examples of −I substituents which can be used in thereagent according to the invention are, for example, halogens. Chlorineand fluorine are well suited. Fluorine is preferred.

[0023] In an alternative form of the invention, everything else beingequal, the reagent is based on an enantiopure amino acid in which atleast one amino group of the amino acid carries an activating group inorder to form an active precursor of an isothiocyanate group. Use may bemade, for example, as activating group, of a thiocarbonyl group bondedto an electronegative group as described above. A(4-nitrophenyloxy)thiocarbonyl or (4-fluorophenyloxy)thiocarbonyl groupis preferred.

[0024] It has been found that, in this alternative form of theinvention, the reaction of the reagent with the organic compoundcomprising a free functional group generally gives rise to the formationof derivatives comprising a thiocarbonyl group which can give aseparation of enantiomers which is further improved with respect to theoxygen-comprising derivatives. The presence of sulphur in thediastereomeric derivatives further facilitates the detection of the saidderivatives, in particular by UV spectrometry.

[0025] In the reagent according to the invention, at least one carboxylgroup of the amino acid is substituted.

[0026] The substituent by which the carboxyl group of the amino acid issubstituted generally does not comprise a free functional group capableof reacting with the active precursor. The substituents which can beused include, for example, linear or branched alkyl groups comprisingfrom 1 to 4 carbon atoms, such as the methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl or t-butyl group, ethers or aryl groups,the alkyl, ethyl or aryl groups optionally being functionalized by, forexample, halogens, carboxylic or sulphonic esters, sulphate esters,phosphonic esters or phosphate esters. Hydrophilic substituents whichensure good solubility of the reagent in mixtures of water with organicsolvents are well suited.

[0027] The hydrophilic substituent generally ensures that a solution ofthe reagent in a 1:1 by volume dioxane/water mixture is homogeneous at20° C. when the concentration of the reagent is at least 0.5×10⁻³ mol/l.The concentration is often at least 1×10⁻³ mol/l. The concentration ispreferably at least 0.5×10⁻² mol/l. Good hydrophilic substituents ensurethat the solution is homogeneous at 20° C. when the concentration of thereagent is at least 1×10⁻² mol/l.

[0028] It is preferable to employ a substituent which comprises at leastone ether bond. Examples of substituents comprising at least one etherbond are, for example, alkyl or aryl ethers of mono-, oligo- orpolyalkylene glycols, such as, for example, mono-, oligo- orpolyethylene glycol or mono-, oligo- or polypropylene glycol. The2-methoxyethyl substituent is particularly preferred.

[0029] Particularly preferred alternative forms of the reagent accordingto the invention comprising a hydrophilic substituent correspond to thegeneral formula (I)

[0030] in which Z₁ and/or Z₂=NO₂, R₁=phenyl, α- or β-indolyl, 1-naphthylor 2-naphthyl, R₂=Me, Et, C₃-C₆ alkyl or C₃-C₆ cycloalkyl, and xrepresents an integer from 1 to 5.

[0031] In an alternative form, the substituent by which the carboxylgroup or the amino acid is substituted comprises at least onechromophore. The term “chromophore” is understood to denote a functionalgroup which absorbs electromagnetic radiation. The absorption maximum ofthe chromophores is generally from 170 to 2500 nm. The absorptionmaximum of the chromophores is preferably from 200 to 1000 nm. Examplesof chromophores which can be used are aromatic systems optionallysubstituted in the 2 or 4 position by an −I, −M substituent as describedabove. The 4-nitrobenzyl, (2-anthraquinonyl)methyl and(9-(9H-fluorenylmethyl)) groups are particularly preferred among thesubstituents comprising at least one chromophore.

[0032] Preferred alternative forms of the reagent according to theinvention comprising at least one chromophore correspond to the generalformula (II)

[0033] in which Z₁ and/or Z₂=NO₂, R₁=phenyl, α- or β-indolyl, 1-naphthylor 2-naphthyl and Y corresponds to any one of the formulae (III to V),the carbon by which Y is bonded to the oxygen of the carboxyl group ofthe enantiopure amino acid being marked by *.

[0034] Use may be made of a hydrophilic substituent comprising at leastone chromophore.

[0035] Particularly preferred alternative forms of the reagent accordingto the invention comprising an active precursor of an isothiocyanategroup and a hydrophilic substituent correspond to the general formula(VI)

[0036] in which Z₁ and/or Z₂=NO₂ or F, R₁=phenyl, α- or β-indolyl,1-naphthyl or 2-naphthyl, R₂=Me, Et, C₃-C₆ alkyl or C₃-C₆ cycloalkyl andx represents an integer from 1 to 5.

[0037] Preferred alternative forms of the reagent according to theinvention comprising an active precursor of an isothiocyanate group andat least one chromophore correspond to the general formula (VII)

[0038] in which Z₁ and/or Z₂=NO₂, R₁=phenyl, α- or β-indolyl, 1-naphthylor 2-naphthyl and Y corresponds to any one of the formulae (III to V),the carbon by which Y is bonded to the oxygen of the carboxyl group ofthe enantiopure amino acid being marked by *.

[0039] When the enantiopure amino acid comprises more than one carboxylgroup, it is preferable to protect all the carboxyl groups. In aparticularly preferred way, all the carboxyl groups are substituted by asubstituent as described above.

[0040] When free functional groups are present in the enantiopure aminoacid, it is preferable to protect the said groups by techniques known inthemselves.

[0041] The reagent according to the invention can be obtained from therespective enantiopure amino acid. Known methods can be used to carryout the esterification of at least one carboxyl group of the amino acid.It is possible, for example, to employ the enantiopure amino acid andthe alcohol corresponding to the substituent to be introduced in anorganic solvent, such as, for example, toluene or benzene, in thepresence of p-toluenesulphonic acid, preferably under azeotropicesterification conditions. Amino acid ester ammonium tosylatederivatives which are well suited to the introduction of an activatinggroup in order to form an active precursor of an isocyanate group areobtained by this synthetic route.

[0042] Mention is made, as example of the introduction of an activatinggroup, of the reaction of an aryloxycarbonyl chloride with the —NH₂group, optionally converted to the ammonium derivative, of an amino acidor of an amino acid ester in basic or neutral medium in a polar organicsolvent. Tertiary amine bases, such as, for example, triethylamine orpyridine, are suitable in particular as base. When the operation iscarried out in neutral medium, it is preferable to employ sodiumhydrogencarbonate. Thus, good results are obtained when an amino acidester ammonium tosylate is reacted with p-nitrophenyloxycarbonylchloride in the presence of sodium hydrogencarbonate in a polar organicsolvent, such as acetonitrile.

[0043] In the process according to the invention, the reagent accordingto the invention is reacted in basic medium with a mixture comprising atleast enantiomers comprising at least one free functional group.

[0044] The reaction of a reagent according to the invention, obtainedfrom the 2-methoxyethyl ester of L-phenylalanine and from 4-nitrophenylchloroformate, with an amino acid is illustrated in a non-limiting wayin Scheme 1 below. The products of this reaction are ureas comprisingtwo amino acids, in which at least one carboxyl functional group issubstituted with a substituent.

[0045] Generally, the free functional group is an optionallymonoalkylated amino group, a hydroxyl group or a thiol group. The freefunctional group can also be composed of an anion, such as, for example,a carbanion or an enolate. The enantiomers comprising at least one freefunctional group which can be separated by the process according to theinvention are generally amino acids, primary or secondary amines,peptides, alcohols, hydroxy acids or thiols. The process according tothe invention gives good results in separating the enantiomers of aminoacids, such as, for example, the amino acids of natural or sytheticorigin mentioned above.

[0046] The process according to the invention also gives good results inseparating a mixture of enantiomers of imino acids. The term “iminoacid” is understood to denote any compound comprising at least one NHRgroup, in which R represents an organic radical, such as, for example,an alkyl or aryl radical, and at least one carboxyl group. Such iminoacids are, for example, those belonging to the group composed ofproline, pipecolic acid (piperidine-2-carboxylic acid),morpholine-3-carboxylic acid, piperazine-2-carboxylic acid,1-thia-4-azacyclohexane-3-carboxylic acid, α-methylproline,cis-4-hydroxyproline, baikaine (1,2,3,5-tetrahydropyridine-2-carboxylicacid), cis-4-hydroxypipecolic acid, trans-5-hydroxypipecolic acid,1,2,3,4-tetrahydronorharman-1-carboxylic acid,1,2,3,4-tetrahydro-6-hydroxyisoquinoline-3-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and N-methylvaline.

[0047] The process according to the invention is carried out in asolvent system in which the mixture of enantiomers and the reagentpossess sufficient solubility and the free functional group possessessufficient nucleophilicity to react with an isocyanate. Systemscomprising at least one polar organic solvent and optionally water aresuitable, for example, as solvent system. Polar organic solvents whichcan be used are, for example, aliphatic or alicyclic ethers, such asdiethyl ether, tetrahydrofuran or 1,4-dioxane, as well as aliphaticesters, such as, for example, ethyl acetate, aliphatic secondary amides,such as, for example, dimethylformamide and dimethylacetamide or, forexample, N-methylpyrrolidone, or acetonitrile.

[0048] Good results for organic compounds comprising a free functionalgroup, such as an optionally monoalkylated amino group, an arylichydroxyl group or a thiol group, are obtained in a solvent systemcomprising a polar organic solvent and water. Dioxane is preferred aspolar organic solvent. The ratio by weight of the polar organic solventto the water in the solvent system is generally less than or equal to99:1. The ratio is most often less than or equal to 75:25. The ratio isgenerally greater than or equal to 1:99. The ratio is most often greaterthan or equal to 25:75.

[0049] The invention also relates to a solution of the reagent accordingto the invention in a polar organic solvent, such as, for example, thepolar organic solvents described above. The concentration of the reagentin the solution is generally at least 1×10⁻³ mol/l. The concentration ispreferably at least 1×10⁻² mol/l. The concentration of the reagent inthe solution is generally at most 1×10⁻¹ mol/l. The concentration ispreferably at most 6×10⁻² mol/l. In the solution according to theinvention, it is preferable to use a polar organic solvent of analyticalpurity. If desirable, the solution according to the invention cancomprise additives, such as, for example, stabilizers.

[0050] The invention also relates to the use of the solution accordingto the invention in an automatic device for the derivatization andseparation of enantiomers of organic compounds comprising at least onefree functional group. The amount of the reagent to be employed dependson the number of free functional groups in the organic compound. Atleast 1 molar equivalent of reagent is employed per free functionalgroup. Generally, at most 10 molar equivalents of reagent are employedper free functional group. Most often, at least 5 molar equivalents ofreagent are employed per free functional group. Preferably, at most 3molar equivalents of reagent are employed per free functional group. Ina particularly preferred way, from 1.1 to 2.5 molar equivalents ofreagent are employed per free functional group.

[0051] The basic nature of the reaction mixture is generated by knownmethods. The operation is preferably carried out in the presence of atleast one base. Tertiary amine bases, such as, for example,triethylamine or diisopropylethylamine, which comprise one basicfunctionality respectively, or N,N,N′,N′-tetramethylethylenediamine,which comprises 2 basic functionalities, are suitable in particular asbase.

[0052] The amount of base to be employed depends on the amount of thereagent and on the number of basic functionalities in the base. Themolar ratio of the reagent to the basic functionalities is generally atleast 1. The ratio is generally at most 2. A ratio of 1 gives goodresults.

[0053] In the process according to the invention, the period of timeduring which the reagent is reacted with the mixture comprising theenantiomers is generally less than or equal to 30 min. Most often, theperiod of time is less than or equal to 20 min. Preferably, the periodof time is less than or equal to 15 min. Good results are obtained witha period of time of greater than or equal to 15 seconds. In practice, aperiod of time of greater than or equal to 1 min is most often applied.A period of time of 5 to 15 min is highly suitable.

[0054] The temperature at which the reagent is reacted with the mixturecomprising at least the enantiomers of an organic compound is generallyless than or equal to 35° C. The temperature is most often less than orequal to 30° C. The temperature is generally greater than or equal to−20° C. The temperature is most often greater than or equal to 0° C. Ina particularly preferred way, the temperature is room temperature, thatis to say generally from 15 to 30° C., preferably 20 to 25° C.

[0055] In the process according to the invention, the mixture ofdiastereomers obtained is subjected to a separation operation. Theseparation operations which can be used for the separation of a mixtureof diastereomers are described, for example, in E. Eliel,Stereochemistry of Organic Compounds, 1994, p. 344-381. Mention may bemade, as examples, of distillation, crystallization and gas or liquidchromatography operations. Among these operations, liquid chromatographyoperations, such as, for example, HPLC chromatography, are preferred. Ina particularly preferred way, the separation operation is RP-HPLC(reverse phase) chromatography. Information regarding HPLCchromatography is found, for example, in Rompp Chemie-Lexikon, 9th Ed.,p. 1860-1861. Use may also be made of thin layer chromatography.

[0056] Eluents which can be used in a chromatography operation areknown. In the case where the process according to the inventioncomprises RP-HPLC chromatography as separation operation, good resultshave been obtained with an eluent comprising acetonitrile or methanol.

[0057] In an alternative form of the process according to the invention,which is preferred, the mixture of diastereomers obtained is subjectedto the separation operation without prior purification. In known methodsfor the separation of enantiomers, a crude mixture of diastereomers isisolated, which mixture has to be subjected to purification prior to theseparation of the diastereomers. The process and the reagent accordingto the invention make it possible not to isolate the crude mixture ofdiastereomers and to carry out the separation operation without priorpurification.

[0058] The process and the reagent according to the invention can beused for the preparative or analytical separation of enantiomers. Theprocess and the reagents are well suited to the analytical separation ofenantiomers. In an alternative form, the process and the reagent areused to determine the enantiomeric excess of an amino acid or of aprimary or secondary amine. In another alternative form, the process andthe reagent are used to determine the enantiomeric excess of a peptide.

[0059] The invention also relates to a process for the production of anenantiopure compound comprising at least one free functional group inwhich:

[0060] (a) a mixture comprising the enantiomers of the compoundcomprising at least one free functional group is subjected to theseparation process according to the invention

[0061] (b) a cleavage operation is carried out on a pure diastereomerobtained by separation of the mixture of diastereomers

[0062] (c) the enantiopure compound is recovered.

[0063] Use may be made, as cleavage operation, of, for example, anoperation of hydrazinolysis in a solvent, such as, for example, analcohol.

[0064] When the process and the reagent according to the invention areused for the analytical separation of enantiomers, use is made of adetection technique known in itself for the determination of the contentof enantiomers in the mixture. Optical techniques, such as, for example,UV spectrometry, visible spectrometry or fluorimetry are highly suitableas detection technique.

[0065] The examples below are intended to illustrate the inventionwithout, however, limiting it.

EXAMPLE 1 Synthesis of the Reagent According to the Invention

[0066] Stage A

[0067] 1 equivalent of enantiopure amino acid, 1.5 equivalents ofpara-toluenesulphonic acid monohydrate, 50 equivalents of toluene PA and5 equivalents of methoxyethanol were introduced into a single-neckedround-bottomed flask. The mixture was heated to reflux and the water wasremoved by means of a Dean and Stark apparatus. After refluxing for 4hours, the reaction mixture was cooled to room temperature and dilutedwith ethyl ether. The diluted reaction mixture was placed in arefrigerator for 16 hours. The white solid obtained was filtered off,washed with ether and dried under vacuum.

[0068] Stage B

[0069] NaHCO₃ (2.6 equivalents) were weighed into a single-neckedround-bottomed flask and acetonitrile was introduced under a stream ofnitrogen (5×10⁻³ mol in 36 ml). The mixture was cooled to 0° C. and4-nitrophenyl chloroformate (1 equivalent), followed by the ammoniumsalt of the enantiopure amino acid obtained according to Example 1,Stage A (1 equivalent), was successively introduced. The mixture wasvigorously stirred for 1 hour at 0° C. and was subsequently brought backto room temperature for 4 hours. At the end of this time, the mixturewas transferred into a separating funnel, was diluted with a 1 molar HClsolution and was extracted three times with ether. The combined organicphases were dried over MgSO₄, filtered and concentrated under reducedpressure. The crude product was subjected to an appropriate purificationtreatment.

EXAMPLE 2

[0070] Stages A and B were carried out by employing L-phenylalanine asenantiopure amino acid. The crude product was recrystallized fromisopropanol until the desired purity was obtained. The yield of Stage Bwas 64%. A crystalline solid was obtained which was stable on storage atroom temperature.

[0071] The reagent thus obtained exhibited the following analyticaldata: NMR (¹H) (dioxane reference at 3.71 ppm; product dissolved ind₆-dioxane) 8.40 (2H, d) 2H of the (4-nitrophenyloxy)carbonyl 7.44 (7H,m) 2H of the (4-nitrophenyloxy)carbonyl and 5H of the phenyl 7.31 (1H,d) NH of the carbamate 4.83 (1H, m) CH of the phenylalanine 4.41 (2H, m)CH₂CO═O 3.70 (2H, m) CH₂OMe 3.47 (3H, s) CH₃O 3.27 (2H, AB) benzyl 2H

[0072] Nuclear magnetic resonance (NMR) was performed, in all cases forwhich an NMR spectrum is shown, with a Brucker AMX 500 MHz devicespectrometer. The chemical shifts are shown in ppm with respect to theresonance of TMS (tetramethylsilane). The following abbreviations areused for the appearance of the resonances: m=multiplet, s=singlet,d=doublet, t=triplet and q=quartet. The number nH indicates the numberof protons corresponding to the signal. Melting point 71-72° C. Opticalrotation [α]: + 42.608 (c = 0.86 g/ 100 ml in dioxane), T° = 26° C., λ =589 nm)

[0073] Thin layer chromatography (TLC) was performed using Merck®60F-254 silica gel plates. Retention factor=0.45 (diethylether/petroleum ether eluent: 75/25 mixture by volume).

EXAMPLE 3-48 Derivatization and Separation of Mixtures of Enantiomers

[0074] The mixture of enantiomers of an organic compound comrising atleast one free functional group according to Examples 3-48 (see Table)(1 equivalent) was weighed accurately in a 10 ml vial and was dissolvedin distilled water (concentration 5×10⁻³ mol/l) in the presence oftriethylamine. The molar ratio of the triethylamine with respect to thereagent was 1. The reagent obtained according to Example 1 in which theenantiopure amino acid was L-phenylalanine was introduced into a second10 ml vial. The reagent was dissolved in dioxane of analytical purity(concentration 3×10⁻² mol/l). The aqueous solution of the amino acid wastransferred via a syringe and with vigorous stirring into the solutionof the derivatization reagent. After stirring for 15 minutes at roomtemperature, 500 μl of the reaction mixture was withdrawn and wasdiluted with 500 μl of dioxane. This solution was analyzed byreverse-phase HPLC by injection of 10 μl of solution onto a Vydac®column.

[0075] The following standard conditions were used for the examples ofthe separation of mixtures of enantiomers by HPLC.

[0076] A Vydac® Reverse Phase C18 CAT 201TP54 column was used.

[0077] An acetonitrile/H₂O mixture with an acetonitrile gradient of1.58%/min in the presence of 0.1% by volume of trifluoroacetic acid wasemployed for the elution.

[0078] Detection was performed by UV spectrometry at 205 and 220 nm.

[0079] The mixtures of enantiomers which were employed with the reagentobtained according to Example 1 and the separation results obtained arecollated in Table 1 below. TABLE 1 Amount of Mixture of reagent^((e))Ex. enantiomers T_(r) L, L^((a)) T_(r) D, L^((b)) α^((c)) R_(s) ^((d))(Equivalents) 3 Alanine 15.08 16.40 1.096 6.01 2 4 Valine 18.73 20.941.127 10.83 2 5 Norvaline 19.28 21.26 1.11 9.45 2 6 Leucine 21.53 23.501.098 8.96 2 7 Norleucine 22.06 23.96 1.091 8.78 2 8 Isoleucine 21.0823.46 1.120 11.55 2 9 Threonine 14.39 15.41 1.078 4.76 2 10Allothreonine 14.39 14.85 1.036 2.09 2 11 Methionine 18.97 20.59 1.0927.74 2 12 13 Glutamic acid 14.56 15.00 1.033 2.07 2 14 Aspartic acid14.22 14.66 1.034 2.08 2 15 Cysteine 26.65 27.19 1.021 2.85  5* 16Cystine 25.59 26.08 1.020 2.76  4* 17 Proline 17.09 17.75 1.042 2.75 218 Phenylalanine 22.64 24.08 1.068 6.25 2 19 Tyrosine 28.82 29.24 1.0151.99  5* 20 Tryptophan 22.79 24.03 1.058 6.54 2 21 Ornithine 24.44 24.921.021 2.26  6* 22 Piperidine-2- 19.86 20.62 1.041 3.51 2 carboxylic acid(pipecolic acid) 23 Morpholine-3- 16.05 15.56 1.034 2.28 2 carboxylicacid 24 1-Thia-4-aza- 18.76 19.43 1.038 3.30 2 cyclohexane- 3-carboxylicacid 25 (2-Naphthyl)- 27.43 28.53 1.042 5.19 2 alanine 26 Homophenyl-24.71 26.20 1.064 7.94 2 alanine 27 (4-Chloro- 25.91 27.02 1.053 5.58 2phenyl)- alanine 28 (4-Fluoro- 23.65 25.033 1.062 6.53 2 phenyl)-alanine 29 (3-Pyridyl)- 14.32 13.57 1.061 3.20 2 alanine 30Phenylglycine 20.98 22.69 1.087 8.24 2 31 2-Methyl- 19.29 20.55 1.075.76 2 proline 32 cis-4- 13.92 14.44 1.041 2.19 2 Hydroxy- praline 33Baikaine 19.05 20.27 1.069 6.03 2 34 cis-4- 15.12 15.91 1.058 3.97 2Hydroxypipe- colic acid 35 trans-5- 13.76 14.36 1.049 2.70 2Hydroxypipe- colic acid 36 2-Amino- 16.84 18.64 1.116 8.31 2 butyricacid 37 1,2,3,4- 23.71 24.75 1.046 4.89 2 Tetrahydro- isoquinoline-3-carboxylic acid 38 1,2,3,4- 23.66^($) 24.67^($) 1.045 4.75 2Tetrahydro- isoquinoline- 1-carboxylic acid 39 erythro-β- 24.03^($)25.73^($) 1.075 8.67 2 Methylphenyl- alanine 40 threo-β- 23.79^($)25.56^($) 1.079 7.22 2 Methylphenyl- alanine 41 o-Methoxy- 23.08^($)24.31^($) 1.057 5.64 2 phenylalanine 42 1,2,3,4- 26.26^($) 27.09^($)1.031 3.44  5* Tetrahydro- norharman-1- carboxylic acid 43 1,2,3,4-29.14^($) 29.67^($) 1.019 2.49  5* Tetrahydro-6- hydroxy- isoquinoline-3-carboxylic acid 44 2-Amino- 18.09^($) 19.21^($) 1.067 6.41 2heptane-1,7- dioic acid 45 erythro-β- 29.84^($) 30.53^($) 1.024 3.65  5*Methyl- tyrosine 46 threo-β- 29.51^($) 30.07^($) 1.02 2.97  5* Methyl-tyrosine 47 N-Methyl- 21.47 22.59 1.056 5.62 2 valine 48 2-Hydroxy-19.79 20.4 1.033 2.73 2 methyl- piperidine Key for Tables 1 to 6^((a))Retention time corresponding to the derivative of the L enantiomer(L, L) ^((b))Retention time corresponding to the derivative of the Denantiomer (D, L) ^((c))Separation factor; α = (T₂2 − T_(r)0) (T₁1 −T_(r)0), where T_(r)2 and T_(r)1 are the retention times of the secondand first compounds respectively and T_(r)0 is the retention time of anon-retained compound. ^((d))Peak resolution:$R_{s} = {0.25\left( \frac{\alpha - 1}{\alpha} \right)\quad \left( \frac{k^{\prime}2}{1 + {k^{\prime}2}} \right)\sqrt{N_{2}}}$

where k′2 is the capacity factor of the second compound and N is thenumber of theoretical plates. *Derivatization also of the functionalgroup carried by the side chain. ^($)The attribution of the retentiontimes to the (L, L) and (D, L) isomers respectively is uncertain as themixture of the enantiomers subjected to the separation was racemic.

EXAMPLE 49

[0080] Stages A and B were carried out by employingL-(2-naphthyl)alanine as enantiopure amino acid. The crude product wasrecrytallized from isopropanol until the desired purity was obtained.The yield of Stage B was 77%. A crystalline solid was obtained which wasstable on storage at room temperature.

[0081] The following NMR spectrum of the reagent was obtained: NMR(¹H)(dioxane reference at 3.71 ppm; product dissolved in d₆-dioxane) 8.20(2H, d) 2H of the (4-nitrophenyloxy)carbonyl 7.79 (4H, m) 4H of thenaphthyl 7.45 (3H, d) 3H of the naphthyl 7.21 (3H, m) 2H of the(4-nitrophenyloxy)carbonyl and 1H of the naphthyl 4.78 (1H, m) CH of thenaphthylalanine 4.27 (2H, m) CH₂CO═O 3.52 (2H, m) CH₂OMe 3.28 (3H, s)CH₃O 3.29 (2H, AB) benzyl 2H Melting point 76-77° C. Optical rotation[α]: 83.5 (c = 1.025 g/100 ml in dioxane, T° = 26° C., λ = 589 nm)

[0082] Thin layer chromatography (TLC) was performed by using Merck®60F-254 silica gel plates. Former retention factor=0.4 (diethylether/petroleum ether eluent: 75/25 mixture by volume).

[0083] The reaction of the 2-methoxyethyl ester ofL-N-(4-nitrophenoxy)carbonyl-(2-naphthyl)alanine with a mixture ofarginine enantiomers and the separation operation was carried out underthe conditions of Examples 3-48. 2 equivalents of reagent and 2equivalents of triethylamine per equivalent of arginine were employed.

[0084] The separation result obtained is shown in Table 2 below. TABLE 2Amount of Mixture of reagent^((e)) Ex. enantiomers T_(r) L, L^((a))T_(r) D, L^((b)) α^((c)) R_(s) ^((d)) (Equivalents) 49 Arginine 20.16819.625 1.030 1.73 2

EXAMPLE 50

[0085] The reaction of the 2-methoxyethyl ester ofL-N-(4-nitrophenoxy)carbonyl-β-tryptophan with a mixture of valineenantiomers and the separation operation were carried out under theconditions of Examples 3-48. 2 equivalents of reagent and 2 equivalentsof triethylamine per equivalent of valine were employed.

[0086] The separation result obtained is shown in Table 3 below. TABLE 3Amount of Mixture of reagent^((e)) Ex. enantiomers T_(r) L, L^((a))T_(r) D, L^((b)) α^((c)) R_(s) ^((d)) (Equivalents) 50 Valine 19.1920.42 1.069 5.80 2

EXAMPLE 51 Preparation of the 4-nitrobenzyl ester ofN-((4-nitrophenoxy)carbonyl)phenylalanine

[0087] Stage A:

[0088] 4 g (1 eq) of Z-(L)-phenylalanine, 2.89 g (1 eq) of 4-nitrobenzylbromide and 26 ml (25 eq) of dimethylformamide were introduced into asingle-necked round-bottomed flask. 1.55 g (2 eq) of dry potassiumfluoride were then added under nitrogen. The mixture was stirred at 60°C. for 16 hours. The reaction mixture was cooled to room temperature anddiluted with 100 ml of ethyl acetate. The organic phase was washed withtwo times 100 ml of a 5% NaHCO₃ solution and with two times 100 ml ofwater. The organic phase was dried over MgSO₄, filtered and concentratedunder reduced pressure. The solid obtained was dried in an ovenovernight.

[0089] Yield: 6.3 g (85%)

[0090] Stage B:

[0091] The solid obtained in Stage 1 was diluted in 25 ml of glacialacetic acid. 6.9 ml (3 eq) of a 33% by weight solution of HBr in aceticacid were carefully added. The mixture was stirred for one and a halfhours at room temperature. The mixture was diluted with 200 ml of ethylether and the white precipitate formed was filtered off and washed withthree times 200 ml of ethyl ether. The solid was dried in an ovenovernight.

[0092] Yield: 5.53 g (99%)

[0093] Stage C:

[0094] 0.82 g of NaHCO₃ (2.6 eq) was weighed in a single-neckedround-bottomed flask and 27 ml of acetonitrile (135 eq) were introducedunder a stream of nitrogen. The mixture was cooled to 0° C. and 0.81 g(1 eq) of 4-nitrophenyl chloroformate, followed by 1.5 g (1 eq) of thebromine salt of the phenylalanine-4-nitrobenzyl derivative, weresuccessively introduced. The mixture was stirred vigorously for 1 hourat 0° C. and was subsequently brought back to room temperature for 11hours. At the end of this time, the mixture was transferred into aseparating funnel, was diluted with 60 ml of a 1M hydrochloric acidsolution and was extracted with three times 60 ml of ethyl acetate. Thecombined organic phases were dried over MgSO₄, filtered and concentratedunder reduced pressure to give a beige solid. The latter was trituratedin 50 ml of ether and filtered off. Yield 1.44 g (82%) NMR(¹H) (methanolreference at 3.32 ppm; product dissolved in d₄-methanol) 8.32 (2H, d) 2Hof the (4-nitrophenyloxy)- carbonyl 8.28 (2H, d) 2H of the4-nitrobenzoyl 7.63 (2H, d) 2H of the 4-nitrophenyoxycarbonyl 7.34-7.41(7H, m) 2H of the 4-nitrobenzyl and 5H of the phenyl 5.37 (2H, s) benzylCH₂ of the 4-nitrobenzyl 4.65 (1H, m) CH of the phenylalanine 3.23 (2H,AB) benzyl CH₂ of the phenylalanine Melting point 118.3° C. Opticalrotation [α]: + 20.83 (c = 0.96 g/100 ml in dioxane, T° = 26° C., λ =589 nm)

[0095] Thin layer chromatography (TLC) was performed by using Merck®60F-254 silica gel plates. Retention factor=0.4 (diethyl ether/petroleumether eluent: 75/25 mixture by volume).

EXAMPLE 52

[0096] The result of the separation of a mixture of valine enantiomerswhich was carried out with the reagent obtained according to Example 51according to the procedure of Example 3 is presented in Table 4.

[0097] Detection was performed by UV spectrometry at 205, 220 and 270nm. TABLE 4 Mixture of Equiva- enanti- T_(r) L, T_(r) D, lents of Ex.omers L^((a)) L^((b)) α^((c)) R_(s) ^((d)) (nm) reagents R_(s) (nm) 52Valine 26.207 27.246 1.042 3.92 2 4.77 (205 nm) (220-270)

[0098] It was observed that this reagent makes possible detection at 220nm and 270 nm. A greater separation factor was obtained at thesewavelengths than when detection was carried out at 205 nm.

EXAMPLE 53

[0099] The 2-methylanthraquinone ester ofN-((4-nitrophenoxy)carbonyl)phenylalanine was synthesized according tothe procedure of Example 51. The result of the separation of a mixtureof valine enantiomers which was carried out with the reagent obtainedaccording to Example 51 according to the procedure of Example 3 ispresented in Table 5.

[0100] The detection was performed by UV spectrometry at 205, 220, 270and 330 nm. TABLE 5 Mixture of Equiva- enanti- T_(r) L, T_(r) D, lentsof Ex. omers L^((a)) L^((b)) α^((c)) R_(s) ^((d)) (nm) reagents R_(s)(nm) 52 Valine 30.168 30.884 1.025 3.01 2 3.11 (205 nm) (270 and 330)

[0101] It was observed that this reagent makes possible detection at 270nm and 330 nm. A slightly greater separation factor was obtained atthese wavelengths than when detection was performed at 205 nm.

[0102] It is apparent that the reagent according to the invention can beeasily obtained. The reagent according to the invention exhibits goodstability at room temperature.

[0103] The process according to the invention makes it possible toseparate a greater variety of chiral organic compounds comprising atleast one free functional group in a simple and fast way and underuniform separation conditions without having to isolate the reactionproduct prior to the separation stage.

EXAMPLE 54

[0104] NaHCO₃ (2.6 equivalents) was weighed in a single-neckedround-bottomed flask and acetonitrile was introduced under a stream ofnitrogen (12×10⁻³ mol in 83 ml). The mixture was cooled to 0° C. and4-fluorophenyl chlorothioformate (1 equivalent), followed by theenantiopure amino acid ammonium salt obtained according to Example 2,Stage A (1 equivalent), were successively introduced. The mixture wasstirred vigorously for 1 hour at 0° C. and was subsequently brought backto room temperature for 4 hours. At the end of this time, the mixturewas transferred into a separating funnel, was diluted with a 1 molar HClsolution and was extracted three times with ether. The combined organicphases were dried over MgSO₄, filtered and concentrated under reducedpressure. The crude product was subjected to an appropriate purificationtreatment.

[0105] The reagent thus obtained was(S)-N-(4-fluorophenoxythiocarbonyl)phenylalanine methoxyethyl ester. Itexhibited the following analytical data: NMR (¹H) (dioxane ref. at 3.71ppm; product dissolved in d₆-dioxane) 8.88 (2H, d) 2H of the4-fluorophenoxythiocarbonyl 7.20-7.49 (7H, m) 2H of thefluorophenoxythiocarbonyl and 5H of the phenyl 6.99 (1H, d) NH of thecarbamate 5.36 (1H, m) CH of the phenylalanine 4.42 (2H, m) CH₂OC═O 3.69(2H, m) CH₂OMe 3.48 (3H, s) CH₃O 3.41 (2H, AB) benzyl 2H

EXAMPLES 55-57

[0106] The derivatization and separation of the mixtures of enantiomersof organic compounds comprising a free functional group of Examples55-57 (Table) were carried out under the conditions of Examples 3-48 byusing, as reagent, the reagent obtained in Example 54. Detection wasperformed only at 245 nm. The separation results obtained are shown inthe table below. TABLE 6 Amount of Mixture of reagent Ex. enantiomersT_(r) L, L^((a)) T_(r) D, L^((b)) α^((c)) R_(s) ^((d)) (Equivalents) 55Valine 24.79 22.11 1.129 12.89 2 56 Proline 19.42 21.22 1.099 7.68 2 57Tyrosine 22.14 23.26 1.054 4.4 5

[0107] The reagent makes possible very efficient separation ofenantiomers. Detection by UV spectrometry can be performed at a singlewavelength and it is highly sensitive.

1-16. (Cancelled)
 17. A reagent based on an enantiopure amino acid inwhich at least one amino group of the amino acid carries an activatinggroup in order to form an active precursor of an isothiocyanate groupand in which at least one carboxyl group of the amino acid issubstituted.
 18. The reagent according to claim 17, in which the reagentis based on an enantipure amino acid selected from the group consistingof alanine, valine, norvaline, leucine, norleucine, isoleucine, serine,isoserine, homoserine, threonine, allothreonine, methionine, ethionine,glutamic acid, aspartic acid, asparagine, cysteine, cystine,phenylalanine, tyrosine, tryptophane, lysine, arginine, histidine,ornithine, glutamine, citrulline, (1-naphthyl)alanine,(2-naphthyl)alanine, homophenylalanine, (4-chlorophenyl)alanine,(4-fluorophenyl)alanine, (3-pyridyl)alanine, phenylglycine,diaminopimelic acid (2,6-diaminoheptane-1,7-dioic acid), 2-aminobutyricacid, 2-aminotetralin-2-carboxylic acid, erythro-β-methylphenylalanine,threo-β-methylphenylalanine, (2-methoxyphenyl)alanine,1-amino-5-hydroxyindan-2-carboxylic acid, 2-aminoheptane-1,7-dioic acid,(2,6-dimethyl-4-hydroxyphenyl)alanine, erythro-β-methyltyrosine andthreo-β-methyltyrosine.
 19. The reagent of claim 17, wherein the aminoacids comprise at least one aromatic nucleus.
 20. The reagent of claim17, wherein said enantiopure amino acid is selected from the groupconsisting of phenylalanine, (1-naphthyl)-alanine, (2-naphthyl)-alanine,α-tryptophan, β-tryptophan, (2-indolyl)alanine and (3-indolyl)-alaninewhich are all optionally substituted.
 21. The reagent of claim 17,wherein the amino acid is phenylalanine.
 22. The reagent of claim 17,wherein said activating group is aryloxycarbonyl, heteroaryloxycarbonyl,1,3-imidazolyly-N-carbonyl or 1,2,4-triazolyl-N-carbonyl group.
 23. Thereagent of claim 22, wherein the aryloxycarbonyl group carries at leastone substituent on the aromatic nucleus wherein said substituent ishalogen, —NO₂, —SO₂R, —SO₂OR, —NR₃ ⁺ or SR₂ ⁺.
 24. The reagent of claim23, wherein the subsitutent is chlorine.
 25. The reagent of claim 23,wherein the substituent is fluorine.
 26. The reagent of claim 17,wherein said activating group is (4-nitrophenyloxy)thiocarbonyl or(4-fluorophenyloxy)thiocarbonyl.
 27. The reagent of claim 17, whereinsaid carboxyl group of the amino acid is substituted by (1) an alkylgroup of 1 to 4 carbon atoms, (2) ether group or (3) aryl group and saidalkyl group, ether group and aryl group are optionally functionalized.28. The reagent of claim 17, said carboxyl group of the amino acid issubstituted by (1) an alkyl selected from the group consisting ofmethyl; ethyl; n-propyl; isopropyl; n-butyl; isobutyl and t-butyl (2)ether; or (3) aryl group, wherein said alkyl, ether and aryl optionallyfunctionalized by halogens, carboxylic esters, sulphonic esters,sulphate esters, phosphonic esters or phosphate esters.
 29. A solutionof the reagent according to claim 17, in a polar organic solvent.